Large-scale genome and proteome projects involve assay techniques that are significantly hindered by current substrate imaging techniques. For example, gel electrophoresis is a critical but slow step in analyses of nucleic acid sequences and proteins. DNA or protein samples arc often electrophoresed in a gel, which separates sample components, and this is followed by formation of an image for quantitation. A gel image can be produced by using a radio-labeled sample and exposing the gel to an x-ray film or storage phosphor plate followed by film development or phosphorescence quantitation. Radioactive labeling, while sensitive, is hazardous and expensive. Fluorescent dyes which bind to the sample and thereupon fluoresce brightly are a preferred method of sample labeling. Application of fluorescence labeling to slab gel electrophoresis and other two-dimensional analysis substrates creates a need for fluorescence imaging instrumentation. Fluorescent intensity images can be created by photographing the substrate or imaging it with a charge coupled device (CCD) areal detector chip. Photography is non-quantitative due to the film's highly non-linear exposure/density function. A CCD detector is capable of quantifying the fluorescence intensity of an image but the spatial resolution is limited by the number of pixels on the chip itself. Both forms of detection involve the use of cameras which have relatively poor collection efficiency, rely on broad and even illumination, and require optics which minimize geometric distortion. These constraints become more acute when imaging large substrates such as electrophoresis gels. Further, cameras are not configured for the rejection of background light, resulting in high background levels.
One alternative for fluorescence quantitation of substrates is image scanning. The fluorescence is measured sequentially at each point in a substrate, creating an image based upon millions of individual pixel measurements. Scanning systems quantify only one point at a time and do not require imaging optics, allowing the optical system to be optimized for collection efficiency and to satisfy other constraints. Fluorescence is typically excited by a laser, which is far brighter and more uniform than other forms of illumination. There are two types of designs for scanning systems and in both types the time required to produce a scanned image increases with the area of the substrate. Scanning-head designs physically translate the excitation and collection optics over the substrate area, resulting in high collection efficiency and noise rejection at the expense of speed. Scanning-beam designs, also commonly termed scanning-spot and flying-spot, accomplish rapid movement of an illumination spot separate from a stationary or slow-moving collection system, imaging rapidly at the expense of collection efficiency.
The designs of existing fluorescence scanning devices provide a trade-off between scanning optic detection systems with high sensitivity and scanning beam detection systems with speed but low sensitivity. Neither system architecture achieves maximum image noise rejection because neither system architecture can eliminate non-random background noise. Background noise consists, in part, of excitation light scattered from the substrate itself as well as from surface and bulk contaminants. Scatter intensity varies with the characteristics of the substrate. Membranes are generally opaque and therefore scatter nearly all the excitation light. Agarose gels are translucent, scattering a fraction of the incident excitation light. Polyacrylamide gels are very clear and exhibit the least scatter. The scattered light component of background noise can consist of elastic scatter at the same wavelength as the incident light and Raman (inelastic) scatter that is red-shifted due to interaction between incident light and vibrating hydrogen-oxygen bonds of water in the substrate. Elastically-scattered light is largely blocked by an emission filter, but it can induce fluorescence in the filter itself which cannot be distinguished from signal fluorescence. Raman scatter often overlaps the emission spectrum of the fluorescent dye, thereby making it past the emission filter. Other significant components of background noise include fluorescence from unbound dyes and autofluorescence of the spectral filter and glass or plastic substrate materials. The foregoing components of background noise are considered to be non-random signals and cannot be removed by conventional signal-averaging techniques.
Therefore, there is a need in the art to provide a high-throughput image scanning device that produces high sensitivity and low background noise fluorescent images, thereby increasing the information content in an image for a given amount of nucleic acid or protein material. Ideally, the device would suppress undesired non-random signals to such a degree that faint fluorescence signals could be imaged even on opaque, scattering, or highly auto-fluorescent substrates.